This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Background &Aims: Identify and characterize the CD4 epitopes that are important not only for the development of effective vaccine for AIDS virus infection but also help understand other diseases pathogenesis. Despite the enormous progress on basic immunology and epitope mapping strategies, the general property of a functional T cell epitope is not yet fully understood. Consequently, an accurate screening strategy to identify the complete epitope repertoire is not available. Moreover, understanding the property of functional T cell epitopes is crucial to understand cellular immunity, mechanisms of viral evasion and will provide a rationale approach for improving AIDS vaccine design. Our hypothesis is that the major characteristic of a functional T cell epitope is to have high binding affinity to the MHC class II proteins as a long peptide fragments during antigen processing. The hypothesis is based on data generated using our novel peptide binding affinity assay using the TCR tetramer for the CTL epitopes. The analysis of five well characterized SIV specific CTL epitopes restricted to the Mamu-A*01 molecule with consistent characteristics and common properties indicate that the TCR tetramer technology can be easily applied to identification of the CD4 T cell epitopes. Finally, since the MHC class II peptide holding groove is wider compared to the MHC class I molecule, we also hypothesize that the number of available CD4 epitopes should be higher than the CTL epitopes. Results: In order to select the appropriate TCR alpha beta sequences for the construction of the TCR tetramer, reassembling of alpha and beta chains from an oligoclonal antigen-specific T cell population into a functional antigen-specific TCR molecule will be necessary. We have optimized the PCR amplification protocol for the TCR alpha beta sequences. With his current protocol, the full length of the TCR alpha and beta sequence could be efficiently amplified from as few as 100 cells following a pre-amplification step. We are now in the process of cloning the full length TCR alpha beta sequences specific for the p46/Mamu-DR*W201 complex. All possible TCR alpha beta combinations will then be co-transfected into Schneider (S2) cells by calcium phosphate precipitation. In order to identify the correct functional TCR alpha/beta pairing, a large amount of the p46/Mamu-DR*W201 tetramer will be needed. Unfortunately, the originally described protein production system using the S2 cells failed to produce the necessary large quantity of stable p46/Mamu-DR*W201 monomers. We are now in the process of developing a new protein expression system adapted from the system used to produce large quantity of recombinant IgG proteins. The Mamu-DR alpha and Mamu-DR*W201 chains have been successfully cloned into the pCIRN Neo-R and pEF IRES GFP vectors, respectively. Both expression vectors will then be co-transfected to 293T cells lines and selected in the presence of neomycin. In order to isolate cells containing both plasmids, the cells will then be sorted for the presence of the GFP protein. Finally, the His-tagged Mamu-DR*W201 proteins present in the supernatant of the large scale culture of the isolated 293T cells will be purified by using the commercially available Ni-column. The positive pair of alpha and beta chains will then be selected to generate the soluble p46 TCR tetramer. Significance The development of an accurate and efficient approach to identify and screen the complete antigen-specific CD4 epitope repertoire restricted to a specific MHC class II molecules in the rhesus monkey model will allow us to better understand antigen-specific CD4 responses to inform development of an efficient vaccine against AIDS.